The synthesis of polymeric biomolecules, such as oligonucleotides, peptides or non-natural analogues thereof, according to the principle of solid phase synthesis is an established technique (2).
Although multiple parallel synthesis of peptides in open reaction systems is part of the prior art (14, 15), oligonucleotides, for example, tend to be prepared individually. The principal problem in oligonucleotide synthesis lies in the extreme sensitivity to water of the phosphoramidite chemistry of the prior art (2). Automated oligosynthesis apparatuses are therefore closed systems and operate under protective gas. A published apparatus for the parallel synthesis of up to 96 oligonucleotides uses a reaction system that likewise is closed and, for the dispensing of reagents, a large number of valves (16). Conventional supports and manual work-up procedures are used therein.
The synthesis system according to the invention is therefore based on the idea of configuring synthesis, supports, anchor groups and work-up procedure for the simultaneous, fully automatic preparation of biomolecules. With the reagents"" being distributed by means of a pipetting robot, the reaction columns can be arranged in a format that is suitable for further processing. In order also to be able to carry out a water- and air-sensitive synthesis protocol using a pipetting robot, it is necessary to take certain measures in terms of design. As an example of a possible solution, there will be described hereinbelow the principle of operation of the automated apparatus and the course of synthesis.
The automated apparatus can operate using conventional supports and reagents, but handling is simplified by specially modified, newly developed supports and anchor groups.
By adding on a special method for simultaneous purification and aliquotting, the quality of the products is improved and use is simplified.
The invention relates especially to the following embodiments: apparatus for automated simultaneous chemical synthesis and purification of a large number of products on solid phase, as well as support material and chemical building blocks for solid phase synthesis, characterised in that
1. a large number (from 10 to 1000, preferably 48 and a multiple thereof, preferably 400) of separate reaction vessels, which are open at the top and at the bottom, are provided in the form of channels or small columns, which are arranged in parallel in a block (FIG. 6) and which are removable either together or separately; the support material for the synthesis (solid phase) is placed in the channels/columns, either being arranged between two inert porous frit plates or, preferably, being itself in the form of a chemically modified frit or filter plate (FIG. 7), so that liquid media added from above are held in the reactor solely as a result of surface tension and wetting of the material;
2. the reactor/reactor block according to 1. is mounted on a trough connected to a vacuum pump by means of a switchable valve, and so the liquid media can be aspirated simultaneously from the reactors and the support materials contained therein;
3. the upper inlets to the reaction columns in the reactor block according to 1., which are covered by a perforated screen (baffle plate) mounted above them, can be flooded with inert gas (e.g. nitrogen, argon) and the flow of inert gas is optionally increased considerably during the aspirating procedure according to 2.; alternatively, the space above the reaction columns/channels can be selectively closed off by means of a second, displaceable perforated screen so that the reagents are blown out of the reaction columns/channels by pressurised inert gas;
4. chemical building blocks, reagents and solvents are distributed to the reaction vessels by an xyz-pipetting robot by means of electronically controllable dispensing syringes (dilutors) having one or more dispensing needles and optionally, in addition, one or more dispensing manifolds so that each reactor can be addressed individually;
5. the dispensing needle according to 4. is equipped with a plurality (at least two) of internal channels, which are connected to separate dispensing syringes, that is to say which can be filled separately, the ends of which channels meet only shortly before the outlet (FIG. 8) and so, when a plurality of reagents are being dispensed simultaneously, mixing occurs in the tip of the dispensing needle shortly before delivery, it being possible for a channel to be connected to the inert gas supply also and so the mixed volume can be expelled by means of a pulse of inert gas;
6. the dispensing needle according to 4. and 5. is mounted so as to be resilient along the longitudinal axis, so that it can set down on the support material or top frits in the reactor channels without damage and so can reliably deposit even extremely small volumes down to 1 nanoliter;
7. a large number (from two to one hundred, preferably 24) of chemical building blocks and reagents, where appropriate dissolved in suitable solvents, are provided in vessels which are sealed by means of septa and which are arranged in a reagent block separate from the reaction block;
8. the septum-sealed necks of the vessels in the reactor block according to 7., which are covered by a perforated screen (baffle plate) mounted above them, can be flooded with inert gas (e.g. nitrogen, argon);
9. using the dispensing needle according to 4. and 5., reagents can also be withdrawn from transfer ports, which are connected to storage bottles either directly or by means of switchable valves (FIG. 6) and the storage bottles are slightly pressurised with inert gas;
10. solvents and reagents can be distributed from solvent bottles by means of dispensing syringes or by pressurised inert gas and also by way of one or more dispensing manifolds according to 4. simultaneously to a plurality of reactors row by row;
11. the support material according to 1. forms a layer in the reactor channel, through which an even flow of the reagents and solvents applied from above passes solely under the action of gravity. According to the principle of solid phase synthesis (FIG. 2), which is part of the prior art, the individual products of each reactor are covalently bonded on the surface of the support material and are built up in parallel in steps by means of a succession of pipetting operations. During all the steps of synthesis (build-up reactions, repetitive protecting group removals and washing operations), the products remain covalently linked to the support material and are removed from the support material, and brought into solution, only in one or more final reaction steps;
12. the chemical building blocks (monomers) used for building up the products are coded as ASCII characters and so the products are described as a sequence of build-up reactions (monomer incorporation reactions) by means of ASCII words; the totality of all products for a synthesis program is consequently a list of ASCII words, which are converted by suitable software on a control computer into valve-switching operations, dispensing-syringe movement operations and robot arm movement operations, it being possible for each monomer incorporation to consist of a succession of several reaction steps and switching operations;
13. linking building blocks (linkers) suitable for covalently linking the products to the support material according to 11. are provided, which allow final removal of the products to take place selectively under mild conditions (FIG. 1). For the synthesis of oligomeric compounds, such as oligonucleotides, peptides etc., it is preferable to provide a xe2x80x9cuniversalxe2x80x9d linker, to which there can also be linked the building blocks of the first build-up reaction of the same chemical reaction type as that used for the further build-up reactions, as a result of which only one type of building block is needed for the entire synthesis of a class of compounds (e.g. only nucleoside-3xe2x80x2-phosphoramidites for the synthesis of 3xe2x80x2-OH-oligonucleotides);
14. in the final removal reactions, the linker according to 13. is preferably firstly converted into a labile, but still intact, form (safety-catch linker), which is then cleaved by means of mild chemical treatment, preferably a pH change. Using a linker of that type, the covalently bonded products can be purified of chemical reagents in a simple manner by means of automatic washing operations on the support material and only at the very end eluted from the reactors into an arrangement of collection vessels which is complementary to the reactor arrangement (FIG. 9);
15. the target products of the syntheses are provided with a group that can be used as an affinity label, by means of which the target products can be bound to a corresponding affinity phase. By that means the products eluted from the reactors are purified in an arrangement of affinity columns which is complementary to the reactor arrangement (FIG. 9). By means of simple automated washing or removal operations, the target products are subsequently eluted from the affinity columns into an arrangement of collection vessels which is complementary to the reactor arrangement;
16. the binding capacities of the affinity columns according to 15. are limited in such a manner that, even when synthesis yields differ, an identical minimum amount of target product is bound and eluted per reactor, so that all the products of a multiple synthesis are obtained in equimolar amounts.
Arranged on the working surface of an exemplary pipetting robot are:
a stand for derivative solutions, optionally under protective gas with septum
reagent withdrawal ports, in this case switchable by valves
reagent bottles under protective gas
a holder for reaction columns.
The reaction columns consist of plastics tubes having inserted frits, which enclose the actual synthesis support and which fix the support in a defined position or have themselves been derivatised as a support. In this instance, commercially available pipette tips have been used as tubes. Alternatively, it is possible to use an injection-moulded component in the form of a unit, comprising individual cavities having filter frits (e.g. from PolyFiltronics, Rockland, Mass., USA).
The upper portion of the reaction system can be flushed with protective gas. A perforated screen in the form of a cover can prevent the ingress of air even when the volume flow is low.
A vacuum can be applied to the lower portion of the reaction system for the purpose of aspirating the reagents. An alternative arrangement uses a second, displaceable perforated screen, by which means the holes can be closed off when necessary and the reaction columns cleared by blowing through pressurised protective gas.
For the purpose of dispensing reagents, the robot is provided with a hollow needle having at least two independent channels. The channels meet only very shortly before the outlet. By means of connected motor-operated dispensing syringes it is possible for reagents to be taken up separately and delivered simultaneously. In the simplest case, the hollow needle consists of two concentrically arranged tubes (see Figure). The hollow needle is mounted so as to be resilient along the longitudinal axis so that even extremely small volumes down to 1 xcexcl can be deposited on the support frits without damaging them.
In order to speed up synthesis, one or more of the reagents and solutions can also be dispensed by means of a distributing manifold. The distributing manifold is either also supplied by a dispensing syringe or it is arranged to be connected, by means of valves, to one or more pressurised storage containers. The distributing manifold dispenses reagents, always simultaneously, to a row of reaction columns. Synthesis then consists of a series of program-controlled transfers of reagents and solvents from the storage containers to the reaction columns. The synthesis procedure and type of reagents are, in principle, known and are part of the prior art.
Synthesis commences with the sequences"" being set out in the form of a list of ASCII character strings in the controlling computer program. The synthesis procedure is defined as a sequence of operations specifying the reagents, the volumes to be transferred in each case and the reaction times to be adhered to. An example of a procedure program is given in the appendix.
The apparatus is then equipped with the necessary reagents and synthesis supports. Conventional supports according to the prior art need to be assigned to the individual sequences, because the first building block has to be attached separately outside the apparatus. A simplification is achieved by using the universal support according to the invention, in which case the first building block is coupled inside the automated synthesis apparatus. The chemistry relating thereto is described in the enclosure.
Synthesis typically begins with a washing step, which is preferably performed by means of the distributing manifold. For the purpose of removing the solvents, the aspirating valve is switched for a specified time and a vacuum is applied to the lower portion of the reaction system. During that time the flow of protective gas to the upper portion is increased considerably in order to substantially prevent the ingress of outside air. Then, typically, a solution for removing the temporary protecting groups, e.g. trichloroacetic acid in acetonitrile, is distributed by means of the hollow needle. The needle is firstly moved into the closed transfer port, which seals tightly against the outside of the needle. The valve is then opened and the reagent is drawn up into one of the channels of the needle. Usually, more than the required amount is drawn up because mixing can occur in the boundary region in the tubing between the needle and dilutor. The boundary region between the liquids is usually defined by an air bubble also drawn up. The valve is re-closed, and the needle moves to the first reaction column. The needle is set down on the top frit or on the support itself, as appropriate, and the first aliquot of reagent is delivered. Movement to the other positions is performed analogously.
The excess in the tubing is then discarded and the hollow needle is rinsed.
In the case of reagents that have to be mixed, for example in order to activate the monomeric building blocks, firstly one of the reagents is drawn up as described. The second (and, where appropriate, further) reagent is then aspirated into the second channel of the hollow needle. In order that the first aliquot delivered will have the correct mixture, some of the excess is discarded at the beginning by actuating both dilutor syringes simultaneously. The needle then moves to the positions specified by the program, sets down on the frit and dispenses the reaction mixture by simultaneously actuating the dilutor syringes. That process is the crucial step in synthesis: by keeping the reagents apart until they are dispensed onto the support, the reaction that competes with the coupling-hydrolysis caused by traces of waterxe2x80x94is delayed. By that means, synthesis in a reaction system that is in fact open to the atmosphere is indeed possible, contrary to the prevailing opinion of those skilled in the art.
The further synthesis procedure consists of a sequence of similar steps using a variety of reagents set out by the program procedure.
Synthesis is followed by the known deprotection and removal of the products from the support, e.g. by incubating with concentrated ammonia solution.
Alternatively, a xe2x80x9csafety-catch linkerxe2x80x9d according to the invention can be used, which remains intact when the protecting groups are removed and is merely brought into a more labile state. All reagents and by-products can therefore be washed out before the synthesis products are removed from the support in a separate step. Using the safety-catch linker makes synthesis work-up considerably easier because ammonia and by-products can be separated off more easily than was previously possible.
Work-up is also made easier as a result of the arrangement of the reaction vessels in a standard format, e.g. that of microtitre plates.
Because the reactions during synthesis never run to completion, it is entirely usual, if not always necessary, to purify the products. For that purpose, the method described by Blxc3x6cker and Frank (9A) can be proposed. Alternatively, it is also possible to use other known affinity purification procedures, e.g. by means of the interaction of biotin and avidin (17A, 17B).
Most applications demand a relatively accurately determined amount of synthesis product. In the case of oligonucleotides for sequencing reactions, that amount is only a fraction of the synthesis yield. In this case the yield for all the synthesis batches can, according to the invention, be made uniform by using a limiting amount of affinity matrix. The amount of affinity matrix is well defined and in each case considerably more labelled synthesis product is applied than corresponds to the binding capacity.
The amounts of synthesis product subsequently eluted are then approximately equal (Attachment 9B). The combination of affinity purification and aliquotting is new and saves elaborate concentration measurements.
As a result of the modular construction of the automated synthesis apparatus, it is possible to combine synthesis, removal, purification and aliquotting in one apparatus. When the method according to EP 0 174 525 is used, an affinity support made from a polymeric material, e.g. polystyrene, polyethylene or modifications thereof, can be used. The material can be so selected that it is inert with regard to the synthesis of protecting group removal. It can then even be arranged as a frit below the synthesis support, which would greatly simplify work-up compared with the prior art.
The working principle of the envisaged multiple automated synthesis apparatus provides for a grid of simple small reactors, in which small porous membrane frits constitute the support material for the synthesis. In each reactor the scale of synthesis should be optimally adapted in line with the application as sequencing primer and be in the range from 1 to 10 nanomol. The concept for chemical derivatisation of the support material for use in the step-wise building-up of oligonucleotides is illustrated in FIG. 1A.
Firstly, detection reactions of adequate sensitivity were established in order to be able to follow the individual derivatisation steps quantitatively on the nanomolar scale:
for hydroxy functions: tritylation with dimethoxytrityl chloride in pyridine followed by acidic detritylation with dichloroacetic acid in dichloroethane and photometric determination of the dimethoxytrityl cation;
for amino functions: staining with the anionic dye bromophenol blue in dimethyl-formamide, followed by basic dissociation and photometric determination of the bromophenol blue anion.
A very wide variety of commercially available polyolefin-based membrane materials were then tested for suitability. Depending on their manufacture, those materials already exhibit non-specific oxidative ageing of the surface with hydroxy, ketone, aldehyde and carboxy groups (FIG. 2). A procedure was therefore developed which firstly xe2x80x9ccleansxe2x80x9d that polymeric surface by reduction and then, by means of selective and readily controllable oxidation, generates exclusively the hydroxy groups required in this case (FIG. 3). By that means it is now possible to produce frit material having a predetermined functionality which is suitable for use for oligonucleotide synthesis. A large number of chemical reactions using those and other hydroxylated materials (e.g. cellulose papers) were carried out in order to attach suitable xe2x80x9cspacersxe2x80x9d and xe2x80x9clinkersxe2x80x9d (FIG. 4). The frits so obtained have been successfully used in a commercial automated synthesis apparatus for oligonucleotide synthesis (FIG. 5) and are now available for tests in the reactor module of the new automated apparatus.
Ideally it should be possible to use the support material (support-spacer-linker), prepared according to FIG. 1A, universally for every oligonucleotide sequence so that individual configuration of the synthesis grid is not necessary. This is not provided by conventional loading in a separate reaction with 3xe2x80x2-nucleoside succinates (each sequence requires one of the four different loaded supports). For that reason, a universal xe2x80x9clinkerxe2x80x9d should be employed, on which oligonucleotide synthesis can be started directly using a first nucleotide building block. The concept of intramolecularly cleavable phosphodiesters according to Kxc3x6ster and Heyns (Tetrahedron Letters 1972, 1531) and Gough et al. (Tetrahedron Letters, 1983, 5321) was utilised for that purpose and suitable linker building blocks were produced and anchored to the support material (FIG. 4, compound type 3). In model syntheses it has been shown successfully that the concept works.
A further aspect of the envisaged synthesis technology is the integrated parallel purification and storage of the oligonucleotides. By using a xe2x80x9csafety-catchxe2x80x9d linkage on the support surface to be developed specifically for that purpose, it will be possible to carry out support-bound deprotection and purification. For that purpose a chemical concept was developed that constitutes a modification of the universal linker (FIG. 4, compound type 3). Corresponding model compounds were prepared, by means of which the feasibility of the concept was checked with a successful outcome.
A first version of the apparatus was constructed on the basis of the original concept, the necessary control software being produced externally (FIGS. 6 to 9).
Depending on the sequence of bases of the synthesised oligonucleotides, different overall yields are obtained. The following method makes it possible to obtain a uniform defined amount of the varous oligonucleotides, the oligonucleotides being purified at the same time.
1. A hydrophobic polyolefin powder (PE, PP, or PTFE) serves as the material for the quantification. Defined amounts of the powder (e.g. 20 mg, 10 mg, 5 mg or 2.5 mg) are introduced into sterile filter tips (Eppendorf) or into appropriate tips fitted with frits. The powder is prepared by washing with acetonitrile (3xc3x97250 xcexcl in each case) and then with 1M triethylamine acetate buffer (TEAA) (3xc3x97250 xcexcl in each case).
2. The oligonucleotides synthesised using the PRIME96 synthesis robot, which oligonucleotides still contain the hydrophobic trityl protecting group, are removed from the support. Solution A is obtained in this manner (in this example: 200 xcexcl of 33% NH3 solution). The resulting solution A is diluted 1:1 with water and applied to the prepared columns and slowly forced through with the aid of a syringe.
3. Rinsing with 2.5% NH3 (aq) (3xc3x97250 xcexcl in each case) and water (3xc3x97250 xcexcl in each case) is carried out.
4. The trityl protecting group is now removed using 2% trifluoroacetic acid (aq) (3xc3x97250 xcexcl in each case). After 5 minutes, rinsing with water (3xc3x97250 xcexcl in each case) is carried out.
5. Quantified and purified oligonucleotide is then eluted using 20% acetonitrile (aq) (3xc3x97250 xcexcl in each case).